New study reveals inner workings of a molecular clamp critical to DNA replication

From bacteria to humans, every organism must replicate its DNA. This basic process, which occurs millions of times a day in an average mammal, is driven by three core protein complexes that act as tiny machines, zipping along an unwound strand of DNA to assemble a duplicate copy. New research from Rockefeller University now shows that one of these complexes, a “clamp loader,” requires several previously unidentified steps to get the process started.

In addition to DNA polymerase, the complex that performs the actual replication of DNA, the duplication process requires a processivity clamp, which keeps the polymerase aligned, and a clamp loader which helps place that clamp on the DNA strand.

In mammals, the clamp is called PCNA, which looks like a doughnut and is made up of three identical proteins. The clamp loader must break open the doughnut in order to load the clamp onto the DNA and alongside the DNA polymerase. The clamp loader is made of five individual proteins linked at four active binding sites. The new research from Mike O’Donnell’s lab, reported this winter in the Journal of Biological Chemistry, has found that each of these four binding sites has a specific, distinct function.

At each site, one protein has a binding pocket for ATP, a molecule involved in energy transfer between components of a cell, and the other protein has an amino acid side chain called an arginine finger. “The arginine finger senses when ATP has bound to the adjacent protein, allowing the clamp loader to load PCNA onto the DNA,” says O’Donnell, head of the Laboratory of DNA Replication and a Howard Hughes Medical Institute investigator. “Later, it sets off a series of events using the energy from the ATPs to ultimately release the clamp loader from PCNA.”

By mutating each of the arginines, both individually and in combination, O’Donnell’s lab showed that each ATP has a specific role. The clamp loader must sense an ATP at one site in order for it to bind both PCNA and DNA. Then, when PCNA is placed around the DNA it sets off a chain reaction at the other sites around the clamp loader to close PCNA around the DNA and eject itself.

“The clamp loader subunit sequences and structures are similar in all single-celled and multi-celled organisms,” says O’Donnell. “So these studies offer insight into one of the most basic and conserved processes known.”

Journal of Biological Chemistry 281(46): 35531-35543 (November 17, 2006)